Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. Only some selected microorganisms are able to transform nitrogen from the abundant gaseous form to usable combined nitrogen compounds. Microorganisms that fix nitrogen are called diazotrophs. Enzymes responsible for nitrogenase action are very susceptible to destruction by oxygen. The nif genes are responsible for the coding of the nitrogenase proteins and other proteins related to and associated with the fixation of atmospheric nitrogen into a form of nitrogen available to plants. The nif genes have both positive and negative regulators. Besides the nitrogenase enzyme, the nif genes also encode a number of regulatory proteins involved in nitrogen fixation. The expression of nif genes is induced as a response to low concentrations of fixed nitrogen and low oxygen concentrations (the low oxygen concentrations are actively maintained in the root environment). In most of the nitrogen fixing microorganisms, activation of transcription of the nif genes is done by the NifA protein. When there is not enough fixed nitrogen available for the use of the nitrogen fixing microorganisms, NifA expression is initiated from the native promoter of the nifA gene, while the NifA protein in turn leads to activation of the remaining nif genes transcription. If there is sufficient amount of reduced nitrogen or if oxygen is present, nifA gene promoter is not activated and no NifA protein expression takes place. On the other hand, another protein, NifL, is activated in presence of reduced nitrogen or oxygen, and this activated NifL inhibits NifA protein activity by interacting with it, resulting in the inhibition of the formation of nitrogenase and all other accessory proteins necessary for nitrogen fixation.
Nitrogen fixation in the free-living, aerobic, heterotrophic, diazotrophic gram negative soil bacterial genus, Azotobacter spp is regulated by the nifLA operon. Here also NifA protein activates the transcription of all the nif genes, while NifL protein antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen. The expression of the nif operons of Azotobacter is mediated by sigma-54 transcription factor, rather than the more common sigma-70 transcription factor. A typical characteristic of sigma 54 transcription factors is the requirement of an activator that must bind to DNA at a site about 100 or more bases upstream of the promoter. The nifA gene is present in Azotobacter in the nifLA operon and is located distal to the promoter of nifLA operon. The nifL gene is also present in Azotobacter in the nifLA operon and is located proximal to the promoter of nifLA operon. Here also, NifL is the negative regulator, which is activated in presence of oxygen or ammonia. NifL inactivates NifA by interacting with it. In addition to NifA, the promoter of the native nifLA operon is also repressed by oxygen and ammonia. The nifLA operon serves as the master regulatory operon for the entire process of nitrogen fixation. Raina et al. [1993, Mol. Gen. Genet. 237: 400-406] has fully characterized the nifL gene of Azotobacter vinelandii and has elucidated its regulation. Unlike in the case of Klebsiella pneumoniae, the expression of the nif LA operon in Azotobacter vinelandii is not autogenously regulated. Bali et al. [1992, App. Env. Microbiol. 58: 1711-1718] inserted an antibiotic resistance cassette upstream of nifA of Azotobacter vinelandii and observed enhanced nitrogen fixation and excretion. Brewin et al. [1999, J. Bacteriol. 181: 7356-7362] studied ammonium excretion in nifL mutants of Azotobacter vinelandii obtained by insertion of antibiotic resistance cassette and concluded that ammonium is excreted from the cell passively. In Azotobacter vinelandii, current evidence suggests that NifL controls the activity of NifA by a relatively stable protein-protein interaction that is modulated by redox changes, ligand binding, and interactions with other signal transduction proteins and membrane components. Azotobacter vinelandii NifL contains a conserved histidine residue found in the transmitter domains of histidine kinases, suggesting that this NifL might employ a classical phosphoryl transfer mechanism to communicate environmental signals to NifA. However, replacement of this conserved histidine by a number of other amino acids does not disable signal transduction. Furthermore, NifL is competent to inhibit NifA in vitro in the absence of ATP, and signal transduction requires stoichiometric protein-protein interactions between the two regulatory proteins (Martinez-Argudo et al., J Bacteriol., 2004, 186(3): 601-610).
In both cereals and non-cereal crops, there is a need to supply extra fixed nitrogen by industrially-fixed nitrogen or biologically fixed nitrogen to supplement nitrogen availability in the soil. Nitrogen released to the available pool by mineralization is expected to depend on the amount of soil-nitrogen removed in the harvested produce, leaching of inorganic nitrogen (e.g. NO3−—N) to groundwater, the magnitude of denitrification of soil-nitrogen as N2O or N2, the extent and duration of immobilization of N and its rate of remobilization in the soil biomass (I. R. Kennedy et al., 2004, Soil Biology & Biochemistry 36: 1229-1244). Inoculant biofertilizers, particularly nitrogen-fixing bacterial diazotrophs, can help ensure that the supply of nutrients contributing to optimized yield is maintained. However, in the presence of chemical nitrogenous fertilizers, there is no biological nitrogen fixation, because the ammonium generated by the chemical fertilizer switches off the nifLA operon and therefore, subsequently all the nif operons.
U.S. Pat. No. 6,548,289 describes a method for increasing the rate of conversion of atmospheric nitrogen into ammonia in the genus Rhizobium, by increasing the intracellular level of the activator protein NifA, by introducing a plasmid containing the nifA gene under an inducible or a constitutive promoter. The problem with this method is that the plasmid has an antibiotic resistance gene, which is undesirable from environmental considerations. Besides, the presence of the antibiotic in the medium or the soil would be essential for stable maintenance of the plasmid inside the bacteria. Thus, continuous selection pressure is required to maintain the stability of the plasmids in the bacteria.
United States Patent Application 20060270555 describes transformation of root nodule bacteria with a catalase gene leading to enhanced nitrogen-fixation ability, a preparation for leguminous crops containing the root nodule bacteria as an active ingredient, and a method of cultivating leguminous crops comprising contacting seeds of crops with the transformed bacteria.
Recombinant microorganisms showing uninterrupted biological nitrogen fixation has been investigated by other researchers by inserting an antibiotic resistance cassette into the nifL gene. However, such microorganisms harboring the antibiotic resistance genes are not acceptable for agricultural use due to environmental concerns. There is a dire need to reduce costs associated with chemical fertilizers and reduce the contribution of these chemical fertilizers to environmental pollution. Therefore, it is highly imperative that novel and inventive products and processes should be developed such that the agricultural costs are reduced with a beneficial effect on the environment.